An experimental model of hypoxia was developed on isolated rat heart to study the effects of hypoxia on cardiac performance and metabolism. Fatty acid (FA) metabolism was explored by external detection with a labelled FA, iodohexadecenoic acid (IHA). Hearts, after 30 min preperfusion in an open system, were transferred in a recirculating system for 40 min and perfused with oleate, glucose, lactate, pyruvate and IHA, either in normoxia (pO2 = 660 mmHg) or in hypoxia (pO2 = 220 mmHg). After 40 min hypoxic recirculation, oxygen uptake and dynamic parameters, except the heart rate, decreased respectively by 56% and 44%, and remained constant throughout the perfusion. Glucose utilization increased 2 fold, endogenous glycogen fell by 50% and lactate + pyruvate production increased 3 fold, showing a stimulation of glycolysis. Oleate uptake decreased by 28%, while triglycerides content remained higher. The ATP/ADP ratio decreased by 24%. Conversely to oleate, IHA uptake was not significantly modified, but its intracellular fate showed a higher radioactivity in all lipid fractions: polar lipids, diglycerides, free FAs and triglycerides. beta oxidation of IHA, evidenced by iodide production, decreased by 39%. The external detection of cardiac radioactivity allowed us to obtain time-activity curves that were analyzed with a 4-compartment mathematical model. The data evidenced an esterification ratio significantly higher in hypoxia. The metabolism of IHA as estimated by the intracellular analysis or, in a non-invasive way, by external detection, was similar to the metabolism of oleate. Thus, lipid metabolism, in hypoxia, can be explored by external detection with IHA.